Detection of Apoptosis by Annexin V PI staining - (Mar/07/2011 )
I have couple of questions regading detection of apoptosis by Annexin V PI staining!
I am inducing apoptosis in B cells under drug treatment and measuring it by Annexin V PI staining using flow cytometer.
I am looking at Annexin V (FL1) positive, PI (FL2) negative population in the dot plot. Under the drug treatment, I see the shift of whole population along the FL1 axis (Annexin V). Hence, I dont have a clear resolution of two different populations under drug treatment. Is it mandatory to have two distinct populations by Annexin V staining or this is absolutely fine considering the shifted part of the population?
If I treat my cells with lower concentration of the drug, I can distinguish two separate populations (double negative & annexin V positive alone). Why should at higher concentration both the populations merge together?
Awaiting to hear from you!!!!!!!!!!!!
Try to titrate you drug. Seems that it is pro-apoptotic.
"...Is it mandatory to have two distinct populations by Annexin V staining..."- the answer is NO.
BTW, do you have all necessary controls (untreated, single stained)?